The Norwalk-like viruses (NLVs) in the family Caliciviridae are the major cause of nonbacterial gastroenteritis. However, the inability to grow these viruses in cell culture has been a continuing research obstacle. The primary purpose of this project is to develop systems for study of the basic molecular biology and replication of these fastidious human pathogens.Efforts continued in the development of an in vitro replication system for the NLVs. We are collaborating with Dr. Ellie Ehrenfeld (NIAID, NIH) and Dr. Craig Cameron (Penn State University) in order to model our in vitro calicivirus replication system after that of poliovirus, a distant, but related positive strand RNA virus. Because the NLVs do not grow in cell culture, we have used the cultivatable feline calicivirus (FCV) to first define the components of the FCV replication complex in infected cells (see Project Z01-AI-00707-05-LID). We have isolated FCV protein complexes from infected cells that actively synthesize RNA in vitro and we are currently identifying the viral (and possibly cellular) proteins associated with these complexes. Upon defining the minimal number of proteins in the FCV replication complex, we will attempt to assemble such a complex for the NLVs. Thus far, we have expressed recombinant proteinase and polymerase (as separate proteins and as a complex) in bacteria for both the Norwalk and Hawaii viruses. The recombinant proteinase from both viruses shows strong proteolytic activity, while the recombinant polymerase has low activity in oligo U-primed Poly A elongation assays. The reason for the low RNA dependent RNA polymerase activity is under investigation, because these studies may give insight into conditions needed for the assembly of an active in vitro replication complex, as well as recovery of the virus from cDNA clones. Of interest, antibodies raised against the Norwalk virus recombinant polymerase recognized the Hawaii virus polymerase in immunoprecipitation assay. The identification of shared antigens between these viruses representing different distinct genetic groups of NLVs may prove useful in the development of diagnostic assays (or vaccines) that are more broadly reactive than those based on the capsid protein.A consensus sequence of the Hawaii virus genome was completed. Sequence analysis of plasmid DNA and PCR products demonstrated that the genome of Hawaii virus is 7,513 bases in length. At the 5?-end of the genome, there are four bases upstream of the first in-frame initiation codon, and 18 nucleotides at the 5? end of the genome that are repeated beginning at position-4 with respect to the first initiation codon of ORF2. The 5? end of ORF2 overlaps the 3? end of ORF1 by 19 nucleotides. The ORF3 initiation codon overlaps the ORF 2 termination codon by one base. The lengths of ORF1, ORF2, and ORF3 are 5097nts, 1605nts and 777nts, respectively. The ORF3 ends in two sequential in-frame stop codons and there is a 39 nucleotide non-coding region between the end of the final stop codon of ORF3 and the beginning of the poly A tract. The length of the poly A tract ranged from 10 to 70 adenine residues when plasmid DNA, containing cloned PCR products spanning the ligation site between 5? and 3? ends of the genome were analyzed, and from 30 to 280 nucleotides when PCR products, obtained using oligo d(T) and an ORF2 specific primer, were sequenced directly. The predicted molecular weight of the polyprotein encoded by ORF1 is 188.9 kiloDaltons (kDa). The predicted molecular weight of the ORF2 gene product (VP1) is 58.7 kDa and is close to the observed molecular weight (57 kDa) for the capsid protein. The predicted molecular weight of the ORF3 gene product (VP2) is 27.3 kDa and is similar to that observed in our previous translation studies of a HV subgenomic template.